Internal Clamp for Surgical Procedures

ABSTRACT

One aspect of the present invention relates to a method of occluding a vascular site in a mammal comprising the step of introducing into the vasculature of a mammal at or proximal to a surgical site, a composition comprising at least one optionally purified inverse thermosensitive polymer, wherein said inverse thermosensitive polymer gels in said vasculature, thereby temporarily occluding a vascular site of said mammal, wherein said temporarily occluded vasculature site is kept in a substantially cylindrical shape.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/793,758, filed Mar. 11, 2013, which is a continuation of U.S.application Ser. No. 12/732,781, filed Mar. 26, 2010, now U.S. Pat. No.8,821,849, which claims priority under 35 U.S.C. §120 to U.S.application Ser. No. 10/983,164, filed Nov. 5, 2004, now U.S. Pat. No.7,700,086, which claims the benefit of U.S. Provisional Application No.60/517,929, filed Nov. 6, 2003 and U.S. Provisional Application No.60/520,888, filed Nov. 18, 2003. The entire teachings of the aboveapplications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Blood vessels are cut during surgical procedures. Electrocautery iseffectively used to reduce or stop hemorrhaging by “burning” thebleeding blood vessels, which seals them off. Various types, shapes, andsizes of tips (probes) are available for specific treatments. A smallelectrode is applied to the skin near the surgical site. This is used tocollect the electricity from the body and safely discharge it back tothe machine. A grounding pad is placed on the person's body (usually thethigh) before the surgery starts to protect the patient. Electrocauteryprevents bleeding from small sized blood vessels and capillary beds.Larger blood vessels require temporary ligation during surgery.

There are two archetypical ways to achieve temporary ligation. The firstway is to ligate from the outside of a blood vessel using clamps, clipsand tourniquets or snares. Such devices press against opposite sides ofa flexible hollow tube so that the walls flatten out and bear againstone another. This produces an axially-extending fold at the two edges.For stopping the flow of fluid through the vessel, this squeezing orpinching action is very effective. However, the lumen of these vesselshave linings (intima), which should not be traumatized by strongdistortions. Strong pressures, and excessive bending (axial folding),can traumatize them leading to complications after the occluder isremoved.

Surgical clamps exist in many sizes with many different types of clampshapes (e.g., curved jaws, straight jaws, etc.). In addition, manydifferent types of jaw surfaces exist, as adapted to the specificfunction performed by the clamp. When a different function is to beperformed, either one must use a different clamp, or in somecircumstances replaceable pads may be added to the jaws. Many existingsurgical clamps have jaws with hard clamping surfaces. Some replaceablepads for these clamps are designed to fit over the jaws to provide asofter clamping surface. Vascular clamps, once they are clamped to theblood vessel, are usually held in the closed position manually by theoperator, or with a locking mechanism.

Clamps and clips have some shortcomings in that atherosclerotic plaquein blood vessels and calcified blood vessels do not withstand thepressure exerted by these devices. It is well known that incrossclamping of the aorta for bypass operations, plaque may be releasedwhen the clamps are opened again and the plaques may lead to strokes(Boivie P, Hansson M, Engstrom K G. “Embolic material generated bymultiple aortic crossclamping: a perfusion model with human cadavericaorta.” J Thorac Cardiovasc Surg 2003 June; 125 (6):1451-60; van derLinden J, Hadjinikolaou L., Bergman P, Lindblom D. “Postoperative strokein cardiac surgery is related to the location and extent ofatherosclerotic disease in the ascending aorta.” J Am Coll Cardiol 2001July; 38 (1):131-5). In addition, especially in older patients,calcified blood vessels, when clamped, may lead to vessel damage.

A second way to achieve temporary ligation is to occlude the blood flowinternally. In temporary ligation using for example balloon angioplasty,a deflated balloon catheter is placed at the arterial or venous site tobe occluded; and then, the balloon is inflated, thereby blocking bloodflow at the site. When the ligation is no longer necessary, the balloonmay be deflated and the catheter removed (Matsuoka S, Uchiyama K, ShimaH, Ohishi S, Nojiri Y, Ogata H. “Temporary percutaneous aortic balloonocclusion to enhance fluid resuscitation prior to definitiveembolization of posttraumatic liver hemorrhage,” Cardiovase InterventRadiol 2001 July-August; 24 (4):274-6; Joseph N, Levy E, Lipman S.“Angioplasty-related iliac artery rupture: treatment by temporaryballoon occlusion.” Cardiovase Intervent Radiol 1987; 10 (5):276-9).However, the inflated balloon leads to dilation of the artery and theinjury to the intima can lead to thickening and narrowing of the artery(Wainwright C L, Miller A M, Wadsworth R M. “Inflammation as a key eventin the development of neointima following vascular balloon injury.” ClinExp Pharmacol Physiol 2001 November; 28 (11):891-5; Labropoulos N,Giannoukas A D, Volteas S K, al Kutoubi A. “Complications of the balloonassisted percutaneous transluminal angioplasty,” J Cardiovasc Surg(Torino) 1994 December; 35 (6):475-89). Another way to internallyocclude blood vessels is a “T” shaped device with a bulbous tip placedat either end of the “T.” These devices are manufactured from siliconrubber. The bulbous tips of the device are inserted into each of the twoparts of the vessel. The bulbous tips have to be the right size toeffectively occlude the blood vessel. Too large and the bulbs willdamage the intima, too small and the bulbs do not efficiently occludeand stop blood flow. See e.g. U.S. Pat. Nos. 3,889,685; 4,168,708;4,946,463. In clinical practice, these devices reduce bleeding at thearteriotomy, but do not stop bleeding. The surgeon, therefore, has stillto rely on additional devices like misted blowers and suction devices toclear the surgical field of blood. Further, the surgeon has to takegreat care not to stitch through the device and be careful duringremoval of the device from the arteriotomy and not entangle the devicein the suture.

Consequently, there is still a need for reversibly stopping blood flowduring surgery, without damage or trauma occasioned by clamps orballoons. This holds great promise in terms of, for example, patientoutcome.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method ofoccluding a vascular site in a mammal, comprising the step ofintroducing into the vasculature of a mammal at or proximal to asurgical site, a composition comprising at least one optionally purifiedinverse thermosensitive polymer, wherein said optional purified inversethermosensitive polymer gels in said vasculature, thereby temporarilyoccluding a vascular site of said mammal.

In certain embodiments, the present invention relates to a method ofoccluding a vascular site in a mammal, comprising the steps ofintroducing into the vasculature of a mammal at or proximal to asurgical site, a composition comprising at least one optionally purifiedinverse thermosensitive polymer, wherein said optional purified inversethermosensitive polymer gels in said vasculature, thereby temporarilyoccluding a vascular site of said mammal; and performing a surgicalprocedure.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of poloxamers and poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpoloxamer 407, poloxamer 338, poloxamer 118, Tetronic® 1107 or Tetronic®1307.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is poloxamer 407.

In certain embodiments, the present invention relates to theaforementioned method, wherein said temporarily occluded vascular site,at or proximal to a surgical site, is a substantially circular orsubstantially elliptical right cylinder, a substantially circular orsubstantially elliptical oblique cylinder, a substantially circular orsubstantially elliptical right truncated cone, or a substantiallycircular or substantially elliptical oblique truncated cone.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of block copolymers, random copolymers, graft polymers,and branched copolymers.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is a polyoxyalkylene block copolymer.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of poloxamers and poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpoloxamer 407, poloxamer 338, poloxamer 118, Tetronic® 1107 or Tetronic®1307.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is poloxamer 407.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of purified poloxamers and purified poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpurified poloxamer 407, purified poloxamer 338, purified poloxamer 118,purified Tetronic® 1107 or purified Tetronic® 1307.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is purified poloxamer 407.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition has a transitiontemperature of between about 10° C. and about 40° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition has a transitiontemperature of between about 15° C. and about 30° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the volume of said composition atphysiological temperature is about 80% to about 120% of its volume belowits transition temperature.

In certain embodiments, the present invention relates to theaforementioned method, wherein the volume of said composition atphysiological temperature is about 80% to about 120% of its volume belowits transition temperature; and said composition has a transitiontemperature of between about 10° C. and about 40° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the volume of said composition atphysiological temperature is about 80% to about 120% of its volume belowits transition temperature; and said composition has a transitiontemperature of between about 15° C. and about 30° C.

In certain embodiments, the present invention relates to theaforementioned method, wherein the volume of said composition atphysiological temperature is about 80% to about 120% of its volume belowits transition temperature; said composition has a transitiontemperature of between about 10° C. and about 40° C.; and saidcomposition comprises at least one optionally purified inversethermosensitive polymer selected from the group consisting of poloxamersand poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein the volume of said composition atphysiological temperature is about 80% to about 120% of its volume belowits transition temperature; said composition has a transitiontemperature of between about 15° C. and about 30° C.; and saidcomposition comprises at least one optionally purified inversethermosensitive polymer selected from the group consisting of poloxamersand poloxamines

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises about 5% toabout 35% of said inverse thermosensitive polymer.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises about 10% toabout 30% of said inverse thermosensitive polymer.

In certain embodiments, the present invention relates to theaforementioned method, wherein the inverse thermosensitive polymer has apolydispersity index from about 1.2 to about 1.0.

In certain embodiments, the present invention relates to theaforementioned method, wherein the inverse thermosensitive polymer has apolydispersity index from about 1.2 to about 1.0.

In certain embodiments, the present invention relates to theaforementioned method, wherein said surgical site is at or proximal to ahemorrhage, cancerous tissue, tumor, or organ.

In certain embodiments, the present invention relates to theaforementioned method, wherein said surgical procedure comprisesanastomosis.

In certain embodiments, the present invention relates to theaforementioned method, wherein said anastomosis comprises connecting afirst vessel and a second vessel.

In certain embodiments, the present invention relates to theaforementioned method, wherein said connecting a first vessel and asecond vessel comprises suturing, laser welding or laser soldering.

In certain embodiments, the present invention relates to theaforementioned method, wherein said anastomosis is selected from thegroup consisting of end-to-end anastomosis, side-to-end anastomosis armside-to-side anastomosis.

In certain embodiments, the present invention relates to theaforementioned method, wherein said occlusion reduces bleeding duringsaid surgical procedure.

In certain embodiments, the present invention relates to theaforementioned method, wherein said occlusion enables controlledischemic preconditioning of said surgical site.

In certain embodiments, the present invention relates to theaforementioned method, wherein said occlusion is at or proximal to anincision site for minimally invasive surgery and decreases bleedingthrough the incision.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition occludes said vascularsite for less than about one hour.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition further comprises acontrast-enhancing agent.

In certain embodiments, the present invention relates to theaforementioned method, wherein said contrast-enhancing agent is selectedfrom the group consisting of radiopaque materials, paramagneticmaterials, heavy atoms, transition metals, lanthanides, actinides, dyes,and radionuclide-containing materials.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition further comprises abiologically active agent.

In certain embodiments, the present invention relates to theaforementioned method, wherein the biologically active agent is selectedfrom the group consisting of antiinflammatories, antibiotics,antimicrobials, chemotherapeutics, antivirals, analgesics,antiproliferatives, plasmids, DNA and RNA.

In certain embodiments, the present invention relates to theaforementioned method, wherein said mammal is a human.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition is introduced to saidvasculature through a percutaneous access device.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition is introduced to saidvasculature using a catheter.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition is introduced to saidvasculature using a syringe.

In certain embodiments, the present invention relates to theaforementioned method, further comprising the step of injecting anaqueous solution at or proximal to the occlusion site, therebydissolving said occlusion.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of poloxamers and poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpoloxamer 407, poloxamer 338, poloxamer 118, Tetronic® 1107 or Tetronic®1307.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is poloxamer 407.

In certain embodiments, the present invention relates to theaforementioned method, further comprising the step of cooling theocclusion site, thereby liquefying the gel and dissolving saidocclusion.

In certain embodiments, the present invention relates to theaforementioned method, said occlusion site is cooled by using a coldaqueous solution or ice.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of poloxamers and poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpoloxamer 407, poloxamer 338, poloxamer 118, Tetronic® 1107 or Tetronic®1307.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is poloxamer 407.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of poloxamers and poloxamines; and said surgicalprocedure comprises anastomosis.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpoloxamer 407, poloxamer 338, poloxamer 118, Tetronic® 1107 or Tetronic®1307; and said surgical procedure comprises anastomosis.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is poloxamer 407; and said surgicalprocedure comprises anastomosis.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition comprises at least oneoptionally purified inverse thermosensitive polymer selected from thegroup consisting of poloxamers and poloxamines.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is selected from the group consisting ofpoloxamer 407, poloxamer 338, poloxamer 118, Tetronic® 1107 or Tetronic®1307.

In certain embodiments, the present invention relates to theaforementioned method, wherein said at least one optionally purifiedinverse thermosensitive polymer is poloxamer 407.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph of viscosity versus temperature for purifiedpoloxamer 407 solutions.

FIG. 2 depicts a graph of blood volume collected from an anastomosissite with and without purified poloxamer 407.

DETAILED DESCRIPTION OF THE INVENTION Definitions

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “anastomosis” as used herein refers to a surgical connectionbetween tubular structures, such as blood vessels. “Beating heart”bypass surgeries, also known as “off-pump” bypass surgeries, areexamples of surgical procedures in which anastomoses are performed.

The term “ischemia” as used herein refers to a lack of blood supply (andthus oxygen) to an organ or tissue.

The term “ischemic preconditioning” as used herein refers to a techniquein which tissue is rendered resistant to the deleterious effects ofprolonged ischemia by prior exposure to brief, repeated periods ofvascular occlusion.

The terms “reversibly gelling” and “inverse thermosensitive” refer tothe property of a polymer wherein gelation takes place upon an increasein temperature, rather than a decrease in temperature.

The term “transition temperature” refers to the temperature ortemperature range at which gelation of an inverse thermosensitivepolymer occurs.

The term, “contrast-enhancing” refers to materials capable of beingmonitored during injection into a mammalian subject by methods formonitoring and detecting such materials, for example by radiography orfluoroscopy. An example of a contrast-enhancing agent is a radiopaquematerial. Contrast-enhancing agents including radiopaque materials maybe either water soluble or water insoluble. Examples of water solubleradiopaque materials include metrizamide, iopamidol, iothalamate sodium,iodomide sodium, and meglumine. Examples of water insoluble radiopaquematerials include metals and metal oxides such as gold, titanium,silver, stainless steel, oxides thereof, aluminum oxide, zirconiumoxide, etc.

As used herein, the term “polymer” means a molecule, formed by thechemical union of two or more oligomer units. The chemical units arenormally linked together by covalent linkages. The two or more combiningunits in a polymer can be all the same, in which case the polymer isreferred to as a homopolymer. They can also be different and, thus, thepolymer will be a combination of the different units. These polymers arereferred to as copolymers.

The term “biocompatible”, as used herein, refers to having the propertyof being biologically compatible by not producing a toxic, injurious, orimmunological response in living tissue.

The term “degradable”, as used herein, refers to having the property ofbreaking down or degrading under certain conditions, e.g. bydissolution.

The term “poloxamer” denotes a symmetrical block copolymer, consistingof a core of PPG polyoxyethylated to both its terminal hydroxyl groups,i.e. conforming to the interchangeable generic formula(PEG)_(X)-(PPG)_(Y)-(PEG)_(X) and (PEO)_(X)-(PPO)_(Y)-(PEO)_(X). Eachpoloxamer name ends with an arbitrary code number, which is related tothe average numerical values of the respective monomer units denoted byX and Y.

The term “poloxamine” denotes a polyalkoxylated symmetrical blockcopolymer of ethylene diamine conforming to the general type[(PEO)_(X)-(PPO)_(Y)]₂-NCH₂CH₂N-[(PPO)_(Y)-(PEO)_(X)]₂. Each Poloxaminename is followed by an arbitrary code number, which is related to theaverage numerical values of the respective monomer units denoted by Xand Y.

The phrase “polydispersity index” refers to the ratio of the “weightaverage molecular weight” to the “number average molecular weight” for aparticular polymer; it reflects the distribution of individual molecularweights in a polymer sample.

Overview

Vascular anastomosis is a procedure by which two blood vessels within apatient are surgically joined together. Vascular anastomosis isperformed during treatment of a variety of conditions including coronaryartery disease, diseases of the great and peripheral vessels, organtransplantation, reconstructive surgery and trauma. In coronary arterydisease (CAD) an occlusion or stenosis in a coronary artery interfereswith blood flow to the heart muscle. Treatment of CAD involves thegrafting of a vessel in the form of a prosthesis or harvested artery orvein to reroute blood flow around the occlusion and restore adequateblood flow to the heart muscle. This treatment is known as coronaryartery bypass grafting (CABG).

In the conventional CABG, a large incision is made in the chest and thesternum is sawed in half to allow access to the heart. In addition, aheart lung machine is used to circulate the patient's blood so that theheart can be stopped and the anastomosis can be performed. During thisprocedure, the aorta is clamped which can lead to trauma of the aortictissue and/or dislodge plaque emboli, both of which increase thelikelihood of neurological complications. In order to minimize thetrauma to the patient induced by conventional CABG, less invasivetechniques have been developed in which the surgery is performed throughsmall incisions in the patients chest with the aid of visualizingscopes. Less invasive CABG can be performed on a beating heart, therebyavoiding the need for a cardiopulmonary bypass.

However, in both conventional and less invasive CABG procedures, thesurgeon has to connect one end of the graft vessel to the coronaryartery and the other end of the graft vessel to a blood supplying veinor artery. This process is a time consuming and difficult procedure andis often further complicated by incomplete occlusion leading to bloodoozing. This is especially problematic in minimally invasive surgerieswhere in the surgeon uses small ports to access the anatomy and roboticsto perform the operation. These procedures often utilize an endoscopiccamera to visualize the surgical field. When this is the case,maintaining a bloodless surgical field is especially important; a singledrop of blood on the camera and the operation might have to be convertedinto a more invasive surgery.

Current medical practice uses a variety of devices to prevent orminimize blood oozing in the surgical practice of anastomosis. Thesimplest device to use would be a clamp, however, the use of clamps inarteriosclerotic vessels is dangerous due to potential dislodgement ofplaque and damage to the artery. Peripherally, tourniquets are oftenused, e.g., in hemodialysis access surgery. A form of tourniquetcommonly used in heart surgery is ligation bands, also called snares,around the artery to be bypassed. If the ligation band is tightened tootightly, damage to the artery might occur; if the ligation band is tooloose, blood is still oozing out of the arteriotomy. Therefore, thesurgeon has to find a compromise between visibility in the surgicalfield and damage to the artery.

Alternatively, an internal vessel occluder device, for example“Flo-Rester” from Synovis, Inc., which is shaped like a Q-Tip, can beinserted into the arteriotomy and mechanically occlude the arteryinternally. The device is extracted just prior to closing theanastomosis. However, the surgeon has to estimate the vessel diametercorrectly for proper fit of the device.

Additionally, a coronary shunt can be deployed. It is shaped similarlyto a Q-Tip, but with a hollow tube for blood flow through the tube.Again, the surgeon has to correctly estimate the diameter of the bloodvessel for proper fit of the device. The shunts and the internal vesseloccluders might be dangerous to deploy in highly arteriosclerotic vesselwith heavy plaque build-up. Furthermore, these devices damage intima dueto mechanical forces on the lining of the blood vessel (see e.g. DemariaR G, Fortier S, Male O, Carrier M, Perrault L P. “Influence ofintracoronary shunt size on coronary endothelial function duringoff-pump coronary artery bypass,” Heart Surg Forum. 6 (2003) 160-8;Hangler H B, Pfaller K, Ruttmann E, Hoefer D, Schachner T, Laufer G,Antretter H. “Effects of intracoronary shunts on coronary endothelialcoating in the human beating heart: Ann Thorac Surg, 77 (2004) 776-80;Demaria R G, Malo, O, Carrier, M, Perrault L P, “The Monoshunt: a newintracoronary shunt design to avoid distal endothelial dysfunctionduring off-pump coronary artery bypass (OPCAB”, Interact CardiovascThorac Surg 2 (2003) 281-286). There are a number of other disadvantagesto these internal vessel occluders: great care has to be taken duringthe anastomosis not to sew in the device as well as not to stitchthrough the device. Further, the removal of these devices is not trivialas great care has to be taken to not entangle the device in the suture.

In one embodiment of the present invention, purified, reversethermosensitive polymers are used to occlude blood vessels duringanastomosis. The purified, reverse thermosensitive polymer solution iseasy to deploy by injection into the arteriotomy utilising a syringeequipped with either a needle or a cannula. As the polymer solutionquickly warms to body temperature and forms the gel plug, it preventsblood oozing due to filling of the artery with the polymer plug andprovides the surgeon a clean, bloodless surgical field. Furthermore, asthe gel fills the artery at the arteriotomy site, the blood vessel iskept in a cylindrical shape; contrary to all other devices used whichresult in flaccid blood vessels due to the emptying of the blood vesselof fluid. As the polymer is highly water-soluble, the polymer plugdissolves in blood and is excreted through the kidney. Cooling thebypass area with sterile ice or cold saline can speed up the dissolutionprocess and enables control of the dissolution time.

In a preferred embodiment, the present invention seeks to replace thecombination of inadequate products and technologies that are used bysurgeons to control blood oozing during anastomosis. In these surgicalprocedures, bleeding is a major difficulty of the procedure. Often it isnot the amount of bleeding that is dangerous for the patient, but any“oozing” makes it difficult for the surgeon to see what he is doing.

Temporarily halting blood flow in the case of hemorrhaging may also beachieved by the instant invention. Often in traumas a surgeon wouldbenefit from halting blood flow for a short period of time to establishfrom where the patient is bleeding. In a preferred embodiment, thepresent invention seeks to halt hemorrhaging by use of a thermosensitivepolymer plug as described herein.

The instant invention may be applied to all surgical procedures, whichinvolve the connection of two blood vessels, e.g., coronary bypass,peripheral bypass, hemodialysis access (creation of a fistula), andfree-flap surgery (breast and face reconstruction surgery). The instantinvention may aid in the establishment of end to end, side to end andside to side anastomoses.

Interestingly, a number of devices, existing or under development, aimat simplifying bypass surgery by automating the sewing of arteries toeach other, or eliminating sewing altogether. They are sometimes calledanastomosis devices. Far from competing with the instant invention,these devices require perfectly cylindrical blood vessels to interactwith. Unlike clamps or snares, which can flatten and damage the artery,the method of the instant invention maintains the cylindrical shape ofthe artery once it is filled with gel, a feature that is very attractiveto the suppliers of anastomosis devices.

Laser welding has been shown to work as an alternative to suturing,however, the process is made more difficult by the need to have acylindrical vessel. Since an artery occluded with gel maintains itscylindrical shape, an additional advantage to the instant occlusionmethod is that alternatives to traditional suturing, such as lasersoldering or welding, may be performed. While various inventors haveproposed devises, e.g., an albumin hollow tube to be inserted into theanastomosis site to keep the artery cylindrical, the instant inventionwill dramatically ease the technical difficulty of these suturingalternatives.

Methods of the Invention

In a preferred embodiment, the present invention significantlysimplifies anastomosis by assuring a bloodless surgical field, obviatesthe surgeon from having to guess the size of the artery at thearteriotomy site, and maintains a substantially cylindrical shape ofsaid artery, by injecting temporary plugs in the vessels being joinedthereby occluding said vessels. The plugs consist of an aqueous solutionof inverse thermosensitive polymers. These polymer solutions are softgels at about 20° C. and as they are injected into the body the gelsfurther stiffens to form hard gels at about body temperature. Thepolymer solution starts externally of the body and thus at a temperaturebelow body temperature.

Introduction/Removal of the Plug

In one embodiment, the polymer solution can be introduced through acatheter. Said catheter can be a dual or multi-lumen catheter. In oneembodiment, the catheter is 3-10 French in size, and more preferably 3-6French.

In another embodiment, a syringe used to inject the inversethermosensitive polymer into the body can be, for example, a 0.1-10 ccsyringe or a syringe with volume of 1-3 cc or with a volume of 0.1-1 cc.Pressure applied to the syringe can be applied by hand or by anautomated syringe pusher (see Example 2 below).

The gelation of reverse thermosensitive polymers is dependent on thetemperature and the concentration of the polymer. Therefore, after theanastomosis procedure, the gel can be removed by instilling a fluidaround the gel, which leads to dissolution of the gel. The fluid may bechilled to help in the dissolution with a preferred temperature of about10° C. below the gelation temperature. The fluid can be instilledthrough a catheter or syringe percutaneously. Alternatively, the site ofthe anastomosis can be chilled by placing sterile ice on the proceduresite, thereby cooling the gel to below its gelation temperature. Theliquid polymer dilutes in blood and is washed away from the anastomosis.

Inverse Thermosensitive Polymers

In general, the inverse thermosensitive polymers used in the methods ofthe invention, which become a gel at or about body temperature, can beinjected into the patient's body in a liquid or soft gel form. Theinjected material once reaching body temperature undergoes a transitionfrom a liquid or soft gel to a hard gel. The inverse thermosensitivepolymers used in connection with the methods of the invention maycomprise a block copolymer with inverse thermal gelation properties. Ingeneral, biocompatible, biodegradable block copolymers that exist as agel at body temperature and a liquid at below body temperature may alsobe used according to the present invention. Also, the inversethermosensitive polymer can include a therapeutic agent such asanti-angiogenic agents, hormones, anesthetics, antimicrobial agents(antibacterial, antifungal, antiviral), anti-inflammatory agents,diagnostic agents, or wound healing agents. Similarly, lowconcentrations of dye (such as methylene blue) or fillers can be addedto the inverse thermosensitive polymer.

The molecular weight of the inverse thermosensitive polymer ispreferably between 1,000 and 50,000, more preferably between 5,000 and35,000. Preferably the polymer is in an aqueous solution. For example,typical aqueous solutions contain about 5% to about 30% polymer,preferably about 10% to about 25%. The molecular weight of a suitableinverse thermosensitive polymer (such as a poloxamer or poloxamine) maybe, for example, between 5,000 and 25,000, and more particularly between7,000 and 20,000.

The pH of the inverse thermosensitive polymer formulation administeredto the mammal is, generally, about 6.0 to about 7.8, which are suitablepH levels for injection into the mammalian body. The pH level may beadjusted by any suitable acid or base, such as hydrochloric acid orsodium hydroxide.

Poloxamers (Pluronics)

Notably, Pluronic® polymers have unique surfactant abilities andextremely low toxicity and immunogenic responses. These products havelow acute oral and dermal toxicity and low potential for causingirritation or sensitization, and the general chronic and sub-chronictoxicity is low. In fact, Pluronic® polymers are among a small number ofsurfactants that have been approved by the FDA for direct use in medicalapplications and as food additives (BASF (1990) Pluronic® & Tetronic®Surfactants, BASF Co., Mount Olive, N. J.). Recently, several Pluronic®polymers have been found to enhance the therapeutic effect of drugs, andthe gene transfer efficiency mediated by adenovirus. (March K L, MadisonJ E, Trapnell B C. “Pharmacokinetics of adenoviral vector-mediated genedelivery to vascular smooth muscle cells: modulation by poloxamer 407and implication for cardiovascular gene therapy” Hum Gene Therapy 1995,6, 41-53).

Poloxamers (or Pluronics), as nonionic surfactants, are widely used indiverse industrial applications. (Nonionic Surfactants: polyoxyalkyleneblock copolymers, Vol. 60, Nace V M, Dekker M (editors), New York, 1996.280 pp.) Their surfactant properties have been useful in detergency,dispersion, stabilization, foaming, and emulsification. (Cabana A,Abdellatif A K, Juhasz J. “Study of the gelation process of polyethyleneoxide, polypropylene oxide-polyethylene oxide copolymer (poloxamer 407)aqueous solutions.” Journal of Colloid and Interface Science. 1997; 190:307-312.) Certain poloxamines, e.g., poloxamine 1307 and 1107, alsodisplay inverse thermosensitivity.

Some of these polymers have been considered for various cardiovascularapplications, as well as in sickle cell anemia. (Maynard C, Swenson R,Paris J A, Martin J S, Hallstrom A P, Cerqueira M D, Weaver W D.Randomized, controlled trial of RheothRx (poloxamer 188) in patientswith suspected acute myocardial infarction. RheothRx in MyocardialInfarction Study Group. Am Heart J. 1998 May; 135 (5 Pt 1): 797-804;O'Keefe J H, Grines C L, DeWood M A, Schaer G L, Browne K, Magorien R D,Kalbfleisch J M, Fletcher W O Jr, Bateman T M, Gibbons R J,Poloxamer-188 as an adjunct to primary percutaneous transluminalcoronary angioplasty for acute myocardial infarction. Am J Cardiol 1996Oct. 1; 78 (7):747-750; and Orringer E P, Casella J F, Ataga K I, KoshyM, Adams-Graves P, Luchtman-Jones L, Wun T, Watanabe M, Shafer F, KutlarA, Abboud M, Steinberg M, Adler B, Swerdlow P, Terregino C, Saccente S,Files B, Ballas S, Brown R, Wojtowicz-Praga S, Grindel J M. Purifiedpoloxamer 188 for treatment of acute vasoocclusive crisis of sickle celldisease: A randomized controlled trial. JAMA. 2001 Nov. 7; 286(17):2099-2106.)

Importantly, several members of this class of polymer, poloxamer 188,poloxamer 407, poloxamer 338, poloxamines 1107 and 1307 show inversethermosensitivity within the physiological temperature range. (Qiu Y,Park K. Environment-sensitive hydrogels for drug delivery. Adv DrugDeliv Rev. 2001 Dec. 31; 53 (3):321-339; and Ron E S, Bromberg L ETemperature-responsive gels and thermogelling polymer matrices forprotein and peptide delivery Adv Drug Deliv Rev. 1998 May 4; 31(3):197-221.) In other words, these polymers are members of a class thatare soluble in aqueous solutions at low temperature, but gel at highertemperatures. Poloxamer 407 is a biocompatiblepolyoxypropylene-polyoxyethylene block copolymer having an averagemolecular weight of about 12,500 and a polyoxypropylene fraction ofabout 30%; poloxamer 188 has an average molecular weight of about 8400and a polyoxypropylene fraction of about 20%; poloxamer 338 has anaverage molecular weight of about 14,600 and a polyoxypropylene fractionof about 20%; poloxamine 1,107 has an average molecular weight of about14,000, poloxamine 1307 has an average molecular weight of about 18,000.Polymers of this type are also referred to as reversibly gelling becausetheir viscosity increases and decreases with an increase and decrease intemperature, respectively. Such reversibly gelling systems are usefulwherever it is desirable to handle a material in a fluid state, butperformance is preferably in a gelled or more viscous state. As notedabove, certain poly(ethyleneoxide)/poly(propyleneoxide) block copolymershave these properties; they are available commercially as Pluronic®poloxamers and Tetronic® poloxamines (BASF, Ludwigshafen, Germany) andgenerically known as poloxamers and poloxamines, respectively. See U.S.Pat. Nos. 4,188,373, 4,478,822 and 4,474,751.

The average molecular weights of the poloxamers range from about 1,000to greater than 16,000 daltons. Because the poloxamers are products of asequential series of reactions, the molecular weights of the individualpoloxamer molecules form a statistical distribution about the averagemolecular weight. In addition, commercially available poloxamers containsubstantial amounts of poly(oxyethylene) homopolymer andpoly(oxyethylene)/poly(oxypropylene diblock polymers. The relativeamounts of these byproducts increase as the molecular weights of thecomponent blocks of the poloxamer increase. Depending upon themanufacturer, these byproducts may constitute from about 15 to about 50%of the total mass of the polymer.

Purification of Inverse Thermosensitive Polymers

The inverse thermosensitive polymers may be purified using a process forthe fractionation of water-soluble polymers, comprising the steps ofdissolving a known amount of the polymer in water, adding a solubleextraction salt to the polymer solution, maintaining the solution at aconstant optimal temperature for a period of time adequate for twodistinct phases to appear, and separating physically the phases.Additionally, the phase containing the polymer fraction of the preferredmolecular weight may be diluted to the original volume with water,extraction salt may be added to achieve the original concentration, andthe separation process repeated as needed until a polymer having anarrower molecular weight distribution than the starting material andoptimal physical characteristics can be recovered.

In certain embodiments, a purified poloxamer or poloxamine has apolydispersity index from about 1.5 to about 1.0. In certainembodiments, a purified poloxamer or poloxamine has a polydispersityindex from about 1.2 to about 1.0.

The aforementioned process consists of forming an aqueous two-phasesystem composed of the polymer and an appropriate salt in water. In sucha system, a soluble salt can be added to a single phase polymer-watersystem to induce phase separation to yield a high salt, low polymerbottom phase, and a low salt, high polymer upper phase. Lower molecularweight polymers partition preferentially into the high salt, low polymerphase. Polymers that can be fractionated using this process includepolyethers, glycols such as poly(ethylene glycol) and poly(ethyleneoxide)s, polyoxyalkylene block copolymers such as poloxamers,poloxamines, and polyoxypropylene/polyoxybutylene copolymers, and otherpolyols, such as polyvinyl alcohol. The average molecular weight ofthese polymers may range from about 800 to greater than 100,000 daltons.See U.S. Pat. No. 6,761,824. The aforementioned purification processinherently exploits the differences in size and polarity, and thereforesolubility, among the poloxamer molecules, the poly(oxyethylene)homopolymer and the poly(oxyethylene)/poly(oxypropylene) diblockbyproducts. The polar fraction of the poloxamer, which generallyincludes the lower molecular weight fraction and the byproducts, isremoved allowing the higher molecular weight fraction of poloxamer to berecovered. The larger molecular weight poloxamer recovered by thismethod has physical characteristics substantially different from thestarting material or commercially available poloxamer including a higheraverage molecular weight, lower polydispersity and a higher viscosity inaqueous solution.

Other purification methods may be used to achieve the desired outcome.For example, WO 92/16484 discloses the use of gel permeationchromatography to isolate a fraction of poloxamer 188 that exhibitsbeneficial biological effects, without causing potentially deleteriousside effects. The copolymer thus obtained had a polydispersity index of1.07 or less, and was substantially saturated. The potentially harmfulside effects were shown to be associated with the low molecular weight,unsaturated portion of the polymer, while the medically beneficialeffects resided in the uniform higher molecular weight material. Othersimilarly improved copolymers were obtained by purifying either thepolyoxypropylene center block during synthesis of the copolymer, or thecopolymer product itself (e.g., U.S. Pat. No. 5,523,492 and U.S. Pat.No. 5,696,298).

Further, a supercritical fluid extraction technique has been used tofractionate a polyoxyalkylene block copolymer as disclosed in U.S. Pat.No. 5,567,859. A purified fraction was obtained, which was composed of afairly uniform polyoxyalkylene block copolymer having a polydispersityof less than 1.17. According to this method, the lower molecular weightfraction was removed in a stream of carbon dioxide maintained at apressure of 2200 pounds per square inch (psi) and a temperature of 40°C.

Additionally, U.S. Pat. No. 5,800,711 discloses a process for thefractionation of polyoxyalkylene block copolymers by the batchwiseremoval of low molecular weight species using a salt extraction andliquid phase separation technique. Poloxamer 407 and poloxamer 188 werefractionated by this method. In each case, a copolymer fraction wasobtained which had a higher average molecular weight and a lowerpolydispersity index as compared to the starting material. However, thechanges in polydispersity index were modest and analysis by gelpermeation chromatography indicated that some low-molecular-weightmaterial remained. The viscosity of aqueous solutions of thefractionated polymers was significantly greater than the viscosity ofthe commercially available polymers at temperatures between 10° C. and37° C., an important property for some medical and drug deliveryapplications. Nevertheless, some of the low molecular weightcontaminants of these polymers are thought to cause deleterious sideeffects when used inside the body, making it especially important thatthey be removed in the fractionation process. As a consequence,polyoxyalkylene block copolymers fractionated by this process are notappropriate for all medical uses.

As mentioned above, the use of these polymers in larger concentrationsin humans requires removal of lower molecular weight contaminantspresent in commercial preparations. As was demonstrated in U.S. Pat. No.5,567,859 (Examples 8 & 9), the lower molecular weight contaminants aremostly responsible for the toxic effects seen. In a clinical trial usingunpurified poloxamer 188, an unacceptable level of transient renaldysfunction was found (Maynard C, Swenson R, Paris J A, Martin J S,Hallstrom A P, Cerqueira M D, Weaver W D. Randomized, controlled trialof RheothRx (poloxamer 188) in patients with suspected acute myocardialinfarction, RheothRx in Myocardial Infarction Study Group. Am Heart J.1998 May; 135 (5 Pt 1):797-804), while another clinical trial usingpurified poloxamer 188 specifically mentioned that no renal dysfunctionwas found (Orringer E F, Casella J F, Ataga K I, Koshy M, Adams-GravesP, Luchtman-Jones L, Wun T, Watanabe M, Shafer F, Kutlar A, Abboud M,Steinberg M, Adler B, Swerdlow P, Terregino C Saccente S, Files B,Ballas S, Brown R, Wojtowicz-Praga S, Grindel J M. Purified poloxamer188 for treatment of acute vasoocclusive crisis of sickle cell disease:A randomized controlled trial. JAMA, 2001 November; 286 (17):2099-2106.)Therefore, it seems imperative to utilize only fractionated poloxamersand poloxamines in vascular applications like the ones envisioned here.Furthermore, fractionation of these thermosensitive polymers leads toimproved gels with stronger mechanical resistance and due to theimproved thermosensitivity requires less polymer to achieve gelation(See for example U.S. Pat. No. 6,761,824 on a purification scheme andthe resultant viscosities).

Anastomosis in Conjunction with Drug Delivery

Effective therapeutic use of many types of biologically active moleculeshas not been achieved simply because methods are not available to causedelivery of therapeutically effective amounts of such substances intothe particular cells of a patient for which treatment would providetherapeutic benefit. Efficient delivery of therapeutically sufficientamounts of such molecules into cells has often proved difficult, if notimpossible, since, for example, the cell membrane presents aselectively-permeable barrier. Additionally, even when biologicallyactive molecules successfully enter targeted cells, they may be degradeddirectly in the cell cytoplasm or even transported to structures in thecell, such as lysosomal compartments, specialized for degradativeprocesses. Thus, both the nature of substances that are allowed to entercells, and the amounts thereof that ultimately arrive at targetedlocations within cells, at which they can provide therapeutic benefit,are strictly limited.

Although such selectivity is generally necessary in order that propercell function can be maintained, it comes with the disadvantage thatmany therapeutically valuable substances (or therapeutically effectiveamounts) are excluded. Additionally, the complex structure, behavior,and environment presented by an intact tissue that is targeted forintracellular delivery of biologically active molecules often interferesubstantially with such delivery, in comparison with the case presentedby populations of cells cultured in vitro. Therefore, new ways ofdelivering drugs at the right time, in a controlled manner, with minimalside effects, and greater efficacy per dose are sought by thedrug-delivery and pharmaceutical industries.

The reversibly gelling polymers used in the anastomosis methods of theinvention have physico-chemical characteristics that make them suitabledelivery vehicles for conventional small-molecule drugs, as well as newmacromolecular (e.g., peptides) drugs or other therapeutic products.Therefore, the composition comprising the thermosensitive polymer mayfurther comprise a pharmaceutic agent selected to provide a pre-selectedpharmaceutic effect. A pharmaceutic effect is one which seeks to treatthe source or symptom of a disease or physical disorder. Pharmaceuticsinclude those products subject to regulation under the FDA pharmaceuticguidelines, as well as consumer products. Importantly, the compositionsused anastomosis methods of the invention are capable of solubilizingand releasing bioactive materials. Solubilization is expected to occuras a result of dissolution in the bulk aqueous phase or by incorporationof the solute in micelles created by the hydrophobic domains of thepoloxamer. Release of the drug would occur through diffusion or networkerosion mechanisms.

Those skilled in the art will appreciate that the compositions used inthe anastomosis methods of the invention may simultaneously be utilizedto deliver a wide variety of pharmaceutic and personal careapplications. To prepare a pharmaceutic composition, an effective amountof pharmaceutically active agent(s), which imparts the desirablepharmaceutic effect is incorporated into the reversibly gellingcomposition used in the anastomosis methods of the invention.Preferably, the selected agent is water soluble, which will readily lenditself to a homogeneous dispersion throughout the reversibly gellingcomposition. It is also preferred that the agent(s) is non-reactive withthe composition. For materials, which are not water soluble, it is alsowithin the scope of the anastomosis methods of the invention to disperseor suspend lipophilic material throughout the composition. Myriadbioactive materials may be delivered using the methods of the presentinvention; the delivered bioactive material includes anesthetics,antimicrobial agents (antibacterial, antifungal, antiviral),anti-inflammatory agents, diagnostic agents, and wound healing agents.

Because the reversibly gelling composition used in the methods of thepresent invention are suited for application under a variety ofphysiological conditions, a wide variety of pharmaceutically activeagents may be incorporated into and administered from the composition.The pharmaceutic agent loaded into the polymer networks of thethermosensitive polymer may be any substance having biological activity,including proteins, polypeptides, polynucleotides, nucleoproteins,polysaccharides, glycoproteins, lipoproteins, and synthetic andbiologically engineered analogs thereof.

A vast number of therapeutic agents may be incorporated in the polymersused in the methods of the present invention. In general, therapeuticagents which may be administered via the methods of the inventioninclude, without limitation: antiinfectives such as antibiotics andantiviral agents; analgesics and analgesic combinations; anorexics;antihelmintics; antiarthritics; antiasthmatic agents; anticonvulsants;antidepressants; antidiuretic agents; antidiarrheals; antihistamines;antiinflammatory agents; antimigraine preparations; antinauseants;antineoplastics; antiparkinsonism drugs; antipruritics; antipsychotics;antipyretics, antispasmodics; anticholinergics; sympathomimetics;xanthine derivatives; cardiovascular preparations including calciumchannel blockers and beta-blockers such as pindolol and antiarrhythmics;antihypertensives; diuretics; vasodilators including general coronary,peripheral and cerebral; central nervous system stimulants; cough andcold preparations, including decongestants; hormones such as estradioland other steroids, including corticosteroids; hypnotics;immunosuppressives; muscle relaxants; parasympatholytics;psychostimulants; sedatives; and tranquilizers; and naturally derived orgenetically engineered proteins, polysaccharides, glycoproteins, orlipoproteins. Suitable pharmaceuticals for parenteral administration arewell known as is exemplified by the Handbook on Injectable Drugs, 6^(th)Edition, by Lawrence A. Trissel, American Society of HospitalPharmacists, Bethesda, Md., 1990 (hereby incorporated by reference).

The pharmaceutically active compound may be any substance havingbiological activity, including proteins, polypeptides, polynucleotides,nucleoproteins, polysaccharides, glycoproteins, lipoproteins, andsynthetic and biologically engineered analogs thereof. The term“protein” is art-recognised and for purposes of this invention alsoencompasses peptides. The proteins or peptides may be any biologicallyactive protein or peptide, naturally occurring or synthetic.

Examples of proteins include antibodies, enzymes, growth hormone andgrowth hormone-releasing hormone, gonadotropin-releasing hormone, andits agonist and antagonist analogues, somatostatin and its analogues,gonadotropins such as luteinizing hormone and follicle-stimulatinghormone, peptide T, thyrocalcitonin, parathyroid hormone, glucagon,vasopressin, oxytocin, angiotensin I and II, bradykinin, kallidin,adrenocorticotropic hormone, thyroid stimulating hormone, insulin,glucagon and the numerous analogues and congeners of the foregoingmolecules. The pharmaceutical agents may be selected from insulin,antigens selected from the group consisting of MMR (mumps, measles andrubella) vaccine, typhoid vaccine, hepatitis A vaccine, hepatitis Bvaccine, herpes simplex virus, bacterial toxoids, cholera toxinB-subunit, influenza vaccine virus, bordetela pertussis virus, vacciniavirus, adenovirus, canary pox, polio vaccine virus, plasmodiumfalciparum, bacillus calmette geurin (BCG), klebsiella pneumoniae, HIVenvelop glycoproteins and cytokins and other agents selected from thegroup consisting of bovine somatropine (sometimes referred to as BST),estrogens, androgens, insulin, growth factors (sometimes referred to asIGF), interleukin I, interleukin II and cytokins. Three such cytokinsare interferon-β, interferon-γ and tuftsin.

Examples of bacterial toxoids that may be incorporated in thecompositions used in the occlusion methods of the invention are tetanus,diphtheria, pseudomonas A, mycobacterium tuberculosis. Examples of thatmay be incorporated in the compositions used in the occlusion methods ofthe invention are HIV envelope glycoproteins, e.g., gp 120 or gp 160,for AIDS vaccines. Examples of anti-ulcer H2 receptor antagonists thatmay be included are ranitidine, cimetidine and famotidine, and otheranti-ulcer drugs are omparazide, cesupride and misoprostol. An exampleof a hypoglycaemic agent is glizipide.

Classes of pharmaceutically active compounds which can be loaded intothat may be incorporated in the compositions used in the occlusionmethods of the invention include, but are not limited to, anti-AIDSsubstances, anti-cancer substances, antibiotics, immunosuppressants(e.g., cyclosporine) anti-viral substances, enzyme inhibitors,neurotoxins, opioids, hypnotics, antihistamines, lubricantstranquilizers, anti-convulsants, muscle relaxants and anti-Parkinsonsubstances, anti-spasmodics and muscle contractants, miotics andanti-cholinergics, anti-glaucoma compounds, anti-parasite and/oranti-protozoal compounds, anti-hypertensives, analgesics, anti-pyreticsand anti-inflammatory agents such as NSAIDs, local anesthetics,ophthalmics, prostaglandins, anti-depressants, anti-psychoticsubstances, anti-emetics, imaging agents, specific targeting agents,neurotransmitters, proteins, cell response modifiers, and vaccines.

Exemplary pharmaceutical agents considered to be particularly suitablefor incorporation in the compositions used in the occlusion methods ofthe invention include but are not limited to imidazoles, such asmiconazole, econazole, terconazole, saperconazole, itraconazole,metronidazole, fluconazole, ketoconazole, and clotrimazole,luteinizing-hormone-releasing hormone (LHRH) and its analogues,nonoxynol-9, a GnRH agonist or antagonist, natural or syntheticprogestrin, such as selected progesterone, 17-hydroxyprogeteronederivatives such as medroxyprogesterone acetate, and 19-nortestosteroneanalogues such as norethindrone, natural or synthetic estrogens,conjugated estrogens, estradiol, estropipate, and ethinyl estradiol,bisphospbonates including etidronate, alendronate, tiludronate,resedronate, clodronate, and pamidronate, calcitonin, parathyroidhormones, carbonic anhydrase inhibitor such as felbamate anddorzolamide, a mast cell stabilizer such as xesterbergsterol-A,lodoxamine, and cromolyn, a prostaglandin inhibitor such as diclofenacand ketorolac, a steroid such as prednisolone, dexamethasone,fluromethylone, rimexolone, and lotepednol, an antihistamine such asantazoline, pheniramine, and histiminase, pilocarpine nitrate, abeta-blocker such as levobunolol and timolol maleate. As will beunderstood by those skilled in the art, two or more pharmaceuticalagents may be combined for specific effects. The necessary amounts ofactive ingredient can be determined by simple experimentation.

By way of example only, any of a number of antibiotics andantimicrobials may be included in the thermosensitive polymers used inthe methods of the invention. Antimicrobial drugs preferred forinclusion in compositions used in the occlusion methods of the inventioninclude salts of lactam drugs, quinolone drugs, ciprofloxacin,norfloxacin, tetracycline, erythromycin, amikacin, triclosan,doxycycline, capreomycin, chlorhexidine, chlortetracycline,oxytetracycline, clindamycin, ethambutol, hexamidine isethionate,metronidazole, pentamidine, gentamicine, kanamycin, lincomycin,methacycline, methenamine, minocycline, neomycin, netilmicin,paromomycin, streptomycin, tobramycin, miconazole and amanfadine and thelike.

By way of example only, in the case of anti-inflammation, non-steroidalanti-inflammatory agents (NSAIDS) may be incorporated in thecompositions used in the occlusion methods of the invention, such aspropionic acid derivatives, acetic acid, fenamic acid derivatives,biphenylcarboxylic acid derivatives, oxicams, including but not limitedto aspirin, acetaminophen, ibuprofen, naproxen, benoxaprofen,flurbiprofen, fenbufen, ketoprofen, indoprofen, pirprofen, carporfen,and bucloxic acid and the like.

Occlusion in Conjunction with Gene Therapy

Another application of the compositions and methods described in theinstant invention would be to aid in the delivery of a wide varietygrowth factors or gene therapeutic agents. Although defective genesassociated with numerous inherited diseases (or that represent diseaserisk factors, including cancer risk factors) have been isolated andcharacterized, methods to correct the disease states themselves, byproviding patients with normal copies of such genes (the technique ofgene therapy), are substantially lacking. By way of example only,diseases that it is hoped may be treated by gene therapy includeinherited disorders such as cystic fibrosis, hemophilias, Gaucher'sdisease, Fabry's disease, and muscular dystrophy (myopathy). Again byway of example, acquired disorders that can be treated include cancer(e.g., multiple myeloma, leukemias, melanomas, ovarian carcinoma andsmall cell lung cancer), cardiovascular conditions (e.g., progressiveheart failure, restenosis), and neurological conditions (e.g., traumaticbrain injury).

Gene therapy requires successful transfection of target cells in apatient. Transfection may generally be defined as the process ofintroducing an expressible natural or synthetic polynucleotide (e.g., agene, a cDNA, or a mRNA) into a cell. Successful expression of theencoding polynucleotide leads to production in the cells of a normalprotein and leads to correction of the disease state associated with theabnormal gene. Therapies based on providing such proteins directly totarget cells (protein replacement therapy) are often ineffective asmethods are not available to cause delivery of therapeutically effectiveamounts of such substances into the particular cells of a patient forwhich treatment would provide therapeutic benefit.

Early in the 1990s, Wolff et al showed that tranfection is achievableusing “naked DNA” injected into muscle (Wolff, J A, et al, Direct GeneTransfer into Mouse Muscle in Vivo”, Science 247 (1990) 1465-1468; Wolffet al, Long-Term persistence of plasmid DNA and foreign gene expressionin mouse muscle”, Hum Mol Genet 1 (1992) 363-369). This transfectionmethod usually leads to a transient expression of the encoded proteins.Furthermore, due to the highly localized nature of the injection, onlylocal areas of gene expression might be achieved. This is highlydisadvantageous in disease like Duchenne's disease, for example, inwhich the whole or large parts of the diaphragm needs to be transfectedto achieve a cure. Further, the transduction levels achieved with nakedDNA are not sufficiently high for therapeutic use.

It was earlier discovered that addition of transfection agents increasedthe transfection rate and therefore, the expression level. There arenumerous transfection agents that have been described in the literature(Felgner P L, Gradek T R, Holm M, Roman R, Chan H W, Wenz M, Northrop JP, Ringold G M, Danielsen M Lipofection: a highly efficient,lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA. 1987November; 84 (21):7413-7. for reviews: Rocha A, Ruiz S, Coll J M,Improvement of DNA transfection with cationic liposomes. J PhysiolBiochem. 2002 March; 58 (1):45-56; Pedroso de Lima M C, Simoes S, PiresP, Faneca H, Duzgunes N., Cationic lipid-DNA complexes in gene delivery:from biophysics to biological applications. Adv Drug Deliv Rev. 2001Apr. 25, 47 (2-3):277-94). Further dramatic improvements in transfectionefficiencies were discovered when using non-ionic polymers incombination with transfection agents (Rolland A P, Mumper R J., Plasmiddelivery to muscle: Recent advances in polymer delivery systems. AdvDrug Deliv Rev, 1998 Mar. 2; 30 (1-3):151-172; Nishikawa, M, Huang, L.,Nonviral vectors in the new millennium: delivery barriers in genetransfer, Hum Gene Ther 20 (2001) 861-70; Ross P C, Hui S W.,Polyethylene glycol enhances lipoplex-cell association and lipofection.Biochim Biophys Acta. 1999 Oct. 15; 1421 (2):273-83). Among non-chargedpolymers discovered to improve the transfection were poloxamers andpoloxamines (Prokop A, Kozlov E, Moore W, Davidson J M., Maximizing thein vivo efficiency of gene transfer by means of nonviral polymeric genedelivery vehicles. J Pharm Sci. 2002 January; 91 (1):67-76; Kabanov A.V, Lemieux P, Vinogradov S, Alakhov V. Pluronic block copolymers: novelfunctional molecules for gene therapy. Adv Drug Deliv Rev. 2002 Feb. 21;54 (2):223-33; Park J S, Oh Y K, Yoon H, Kim J M, Kim C K. In situgelling and mucoadhesive polymer vehicles for controlled intranasaldelivery of plasmid DNA. J Biomed Mater Res. 2002 January; 59(1):144-51).

It has also been noted that physical effects can increase the rate oftransfection. Liu et al demonstrated a simple method of intravasculargene transfection by injection of large volumes of DNA containingsolutions into the tail of mice (Liu F, Song Y, Liu D.Hydrodynamics-based transfection in animals by systemic administrationof plasmid DNA, Gene Ther. 1999 July; 6 (7):1258-66; Jiang J, Yamato E,Miyazaki J. Intravenous delivery of naked plasmid DNA for in vivocytokine expression. Biochem Biophys Res Commun. 2001 Dec. 21; 289(5):1088-92.). They termed this method hydrodyoamic transfection.Presumably the increase in transfection versus control is due to anincrease in pressure in the blood vessels, increasing the permeabilityof the cell walls.

A similar effect was discovered by Song et al when the retention timewas increased in the vasculature (Song Y K, Liu F, Liu D. Enhanced geneexpression in mouse lung by prolonging the retention time ofintravenously injected plasmid DNA. Gene Ther. 1998 November: 5(11):1531-7). The increase in retention was achieved by injecting theliposomes prior to the injection of the naked DNA. As the liposomesincrease in size due aggregation caused by different components inblood, they reach the size of capillaries and temporarily occlude theblood vessels. An increase in DNA retention time in the lung results ina higher level of gene expression in the targeted lung area. Controlsusing naked DNA without prior injection of liposomes did not enhancegene transfer (Barron L G, Uyechi L S, Szoka F C Jr., Cationic lipidsare essential for gene delivery mediated by intravenous administrationof lipoplexes. Gene Ther. 1999 June; 6 (6):1179-83). These resultssuggest that prolonging the exposure time of DNA to the target cells invivo may be an important strategy in achieving a high level of geneexpression.

While Song et al utilized liposomes to increase the DNA retention in thetarget area, Liu and Huang used surgery to reach the target area andutilized clamps to temporarily stop blood flow and increase theresidence time of the DNA in the target vasculature (Liu F, Huang L,improving plasmid DNA-mediated liver gene transfer by prolonging itsretention in the hepatic vasculature. J Gene Med. 2001November-December; 3 (6):569-76). They stopped blood flow for a veryshort time and demonstrated that effective gene transfer occurred.Barron et al also showed that the gene transfer occurs rather rapidlywithin less than 60 minutes after injection of the lipoplexes (Barron LG, Gagne L, Szoka F C Jr. Lipoplex-mediated gene delivery to the lungoccurs within 60 minutes of intravenous administration. Hum. Gene Ther,1999 Jul. 1; 10 (10):1683-94).

However the gene therapy approach described by Liu and Huang (surgicalclamping, prior to injection of liposomes) is not practical in aclinical setting, as it would be preferable to introduce thepolynucleotide via a catheter or a syringe. It is well understood thatliposome-mediated or naked DNA gene transfection can be successfullyperformed to all vessel layers or muscles in vivo by using a localdelivery catheter (Hagstrom J E. Plasmid-based gene delivery to targettissues in vivo: the intravascular approach. Curr Opin Mol Ther. 2003August; 5 (4):338-44). However, unwanted transfection at a distance mayoccur with catheter-based local delivery and therefore a device isneeded to occlude the target area to achieve an increase in retentiontime. While this could be achieved by using a balloon catheter and theinjection through the balloon, balloon angioplasty is known to lead toarterial damage and even rupture of the artery (Wainwright C L, Miller AM, Wadsworth R M, Inflammation as a key event in the development ofneointima following vascular balloon injury. Clin Exp Pharmacol Physiol,2001 November; 28 (11):891-5; Labropoulos N, Giannoukas A D, Volteas SK, al Kutoubi A., Complications of the balloon assisted percutaneoustransluminal angioplasty. Review article. J Cardiovase Surg (Torino).1994 December; 35 (6):475-89).

In a preferred embodiment the methods and compositions of the instantinvention can be used to temporarily occlude a blood vessel, eitherindependently our in conjuction with a surgical procedure such asanastomosis, prior to or concurrently with the introduction of nucleicacids behind the occlusion, thereby increasing the residence time ofnucleic acids in an intravascular target area. The nucleic acids arethen injected through the gel by either a syringe or a catheter and thenucleic acid are retained behind the gel in the target area. If theocclusion is on the venous side, the nucleic acid is injected in thearterial side and as there is no drainage until the gel erodes, thenucleic acids are retained on the arterial side. Alternatively, bothproximal and distal sites from the target area may be occluded with areverse thermosensitive polymer composition of the instant invention. Asdescribed above, the polynucleotide can be “naked” or complexed intolipoplexes constituting nucleic acids, cationic lipids, and optionallyhelper lipids.

In certain embodiments, the present invention relates to theaforementioned method, in which a reverse thermosensitive polymer isinjected into a blood vessel proximally to the target area, in vivo, insuch a way that the mixture gels and temporarily and reversibly occludesthe vascular site and the nucleic acid is injected through the gel intothe stagnant blood of the target area.

In certain embodiments, the present invention relates to theaforementioned method, in which a reverse thermosensitive polymer isinjected into a blood vessel proximally and distally to the target area,in vivo, in such a way that the mixture gels and temporarily andreversibly occludes the vascular site and the nucleic acid is injectedthrough the gel on the proximal side into the stagnant blood of thetarget area.

In certain embodiments, the present invention relates to theaforementioned method, in which a reverse thermosensitive polymer isinjected distally from the target area, in such a way as to occludeblood drainage from the target area and nucleic acids or a nucleic acidcomplex are injected into a blood vessel proximally to the target area.

In certain embodiments, the present invention relates to theaforementioned methods, in which a reverse thermosensitive polymer isinjected into a blood vessel proximally to the target area, in vivo, insuch a way that it gels and temporarily and reversibly occludes thevascular site, and a nucleic acid is injected into the same blood vesselat the same time.

In certain embodiments, the present invention relates to theaforementioned method, in which a reverse thermosensitive polymer isinjected distally from the target area, in such a way as to occludeblood drainage from the target area and nucleic acids or a nucleic acidcomplex are injected in a hypertonic solution into a blood vesselproximally to the target area.

In certain embodiments, the present invention relates to theaforementioned method, in which a reverse thermosensitive polymer isinjected distally from the target area, in such a way as to occludeblood outflow from the target area and nucleic acids or a nucleic acidcomplex are injected hydrodynamically into a blood vessel proximally tothe target area.

In certain embodiments, the present invention relates to theaforementioned method, in which the nucleic acid is injected at the sametime as the occlusion of the blood vessel.

In certain embodiments, the present invention relates to theaforementioned method, in which the nucleic acid is injected within lessthan about one minute after occlusion of the blood vessel.

In certain embodiments, the present invention relates to theaforementioned method, in which the nucleic acid is injected within lessthan about ten minutes after occlusion of the blood vessel.

In certain embodiments, the present invention relates to theaforementioned method, wherein the nucleic acid is a plasmid, cDNA, mRNAand PNA.

In certain embodiments, the present invention relates to theaforementioned method, wherein the nucleic acid is not complexed(“naked”).

In certain embodiments, the present invention relates to theaforementioned method, wherein the nucleic acid is complexed withcationic lipids into lipoplexes.

In certain embodiments, the present invention relates to theaforementioned method, wherein the nucleic acid is complexed withcationic lipids and helper lipids into lipoplexes.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition is introduced into thevasculature of said mammal using a catheter.

In certain embodiments, the present invention relates to theaforementioned method, wherein said composition is introduced into thevasculature of said mammal using a syringe.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1 In-vitro Testing and Principal of Operation

The viscosity changes were measured in a Brookfield Cone and Cupviscometer with temperature control. A graph of the viscosity changes(FIG. 1) clearly shows polymer concentrations from approximately 12.5 w% until at least 20 w % will show steep increases in solutionviscosities with temperature. The onset of gelation is dependent on thetemperature and higher polymer concentrations lead to earlier onsets ofgelation. Furthermore, polymer concentrations below approximately 12.3 w% do not demonstrate an increase in solution viscosity with temperatureand remain liquid even at body temperature.

These two findings demonstrate the potential operation principle of thepurified poloxamer 407. The polymer solution is injected as a soft gelat the temperature of a typical OR (about 18° C.) into the arteriotomyand the rise in temperature leads to a stiff gel. The gel will start todissolve in blood and when the concentration of the polymer decreasesbelow approximately 12.5%, it turns back into a liquid, without anypossibility to turn back into a gel at physiological temperatures.Alternatively, cooling of the gel with ice or cold saline would liquefythe gel as the temperature falls below the gelation point. As a liquid,it quickly dilutes in blood and again there is no possibility for it toturn bank into a gel at physiological temperatures.

Example 2 Injectability of Purified Poloxamer 407 Through Various NeedleGauges

A three milliliter polycarbonate syringe (Merrit Medallion) was loadedin the cold with three milliliter of 20 w % purified poloxamer 407.Various sized needles were attached via a luer lock and theinjectability of the polymer solution was tested at 6° C. (liquid state)and at room temperature (23° C.; soft gel state) as shown in the tablebelow.

TABLE 1 Injectability of 20 w % purified poloxamer 407 through a 3 mLsyringe Needle 6° C. 23° C. 16G easy easy 18G easy easy 21G easy easy25G easy pushable 27G easy required hard push

The same experiment was repeated using a one milliliter polycarbonatesyringe (Merrit Medallion) and in all cases, the polymer could be easilyinjected through the various needle gauges.

TABLE 2 Injectability of 20 w % purified poloxamer 407 through a 1 mLsyringe Needle 6° C. 23° C. 16G easy easy 18G easy easy 21G easy easy25G easy easy 27G easy easy

Example 3 In-Vivo Feasibility Animal Trial: Occlusion Experiments

The experiments described below were conducted in pigs. A segment of thedistal half of the LAD was selected for the experiment. The diameter ofthe LAD varied from about 1 to 2 millimeter. An ultrasound flow probewas placed distally from the injection site around the LAD. A onemilliliter syringe equipped with a 27 G needle was used to injectapproximately 200 μL of 20 w % purified poloxamer 407 into the LAD. Flowstopped immediately as evidenced by the flow probe. The occlusionremained between 6 minutes and 18 minutes, averaging approximately 9minutes. Blood flow was reestablished immediately after the occlusiondissolved, a typical hyperemic response was seen with an approximatedoubling of the blood flow. Blood flow returned to normal values within20 minutes.

Example 4 In-Vivo feasibility Animal Trial: Arteriotomy Experiments

The experiments described below were conducted in three mongrel dogs. Asegment of the distal half of the LAD was selected for the anastomosticsite. Silicone Elastic tapes were passed deep to the LAD approximately 2cm apart, flanking the chosen site. A Genzyme Immobilizer OP-CABstabilizer was positioned to stabilize the LAD and the tapes snared totransiently interrupt coronary flow. The immobilizer was left inposition for 3 minutes to provide pre-ischemic conditioning, so that theanimal would subsequently tolerate longer periods of regional ischemia.After 3 minutes, the tapes were loosened to allow reperfusion. After anadditional 5 minutes of reperfusion, the tapes were snared once again,and the cycle repeated two additional times.

After 3 cycles of pre-ischemic conditioning, the Silicone-Elastic tapeswere snared and the LAD arteriotomy created. In this acute animal model,hemostasis was excellent, given the elastic nature of disease-freecoronaries, and the lack of collaterals in the absence of coronarystenosis or occlusion. Therefore, the proximal tape was loosenedslightly until modest bleeding occurred, approximating the amount ofbleeding seen in difficult anastomosis. About one milliliter of a 20 w %solution of purified poloxamer 407 was instilled into the coronarythrough the arteriotomy via a cannula to improve hemostasis. Bleedingstopped immediately and the surgical field was bloodless. Thearteriotomy was not sutured and the site started rebleeding afterapproximately 17 minutes. The experiment was repeated at the same siteand rebleeding occurred after approximately 19 minutes.

All three dogs underwent the same procedure and could be evaluated. Theoccluded site on the LAD was excised and evaluated by histology. Neitherischemic damage nor early necrosis was detected and normal myocardiumwas found.

Example 5 In-Vivo Feasibility Animal Trial: Control of Dissolution Time

The same experiment as described above was performed on the LAD of amongrel dog and approximately 0.6 milliliter of a 20 w % purifiedpoloxamer 407 solution was injected into the arteriotomy via a cannula.Bleeding stopped immediately. The gel kept the artery in a cylindricalshape and the lids of the arteriotomy were clearly visible. Suturing wasperformed through the gel and and was completed within 7 minutes. TheLAD was still occluded and small amounts of sterile ice were placed onthe LAD at the occlusion site to convert the polymer plug back intosolution. The LAD opened up again nearly instantaneously. Thisexperiment demonstrated A) the possibility to control the occlusion timeby using ice placed onto the arteriotomy. This is a very importantfeature as surgeons would want to be able to reopen the occlusion shouldsomething go wrong during the surgery and blood flow is needed. And B)that gel keeps the artery in a cylindrical shape during the occlusion,making suturing very easy.

Example 6 In-Vivo Feasibility Animal Trial: Comparison of Blood Losswith Snares, and Snares & 20 w % Purified Poloxamer 407 Gel

The following experiments were conducted in 4 pigs. The goal of theexperiment was to measure blood loss as a distinction between ligationbands and polymer plug. Further, the patency of the graft was evaluatedby fluoroscopy after utilizing the polymer plug for the anastomosis.

The same approach for OPCAB was used as described above to create theacute animal model with some modifications as described below.

The Immobilizer was modified by attaching a drape to make a shallow wellthat captured all coronary blood and allowed the amount of bleeding fromthe arteriotomy to be quantified.

After 3 cycles of pre-ischemic conditioning, as described above, theSilicone-Elastic tapes were snared and the LAD arteriotomy created. Atthis point, the adequacy of hemostasis was assessed. The proximal tapewas loosened slightly until modest bleeding occurred, comparable to whatis generally encountered clinically.

While maintaining blood pressure, the bleeding was allowed to continuefor 15 minutes to approximate the time required for a distalanastomosis. During these 15 minutes, blood that accumulated in themodified stabilizer well was drawn intermittently through a syringe andmeasured in a volumetric cylinder. After the 15 minutes had passed, ashunt was deployed in the LAD for 20 minutes to allow for reperfusion ofthe myocardium. After reperfusion, the tapes were again tightened tovery similar tension as before, bleeding out of the arteriotomy wasassured and blood pressure was maintained.

Approximately 300 μL of the purified poloxamer 407 at the temperature ofthe OR (about 18° C.) was instilled upstream and approximately 100 μLdownstream into the coronary through the arteriotomy site using acannula. With the polymer gel in place, any blood that accumulated inthe stabilizer basin was removed by syringe. In contrast to the dogexperiments, the arteriotomy opened again between 6.5 and 8.5 minutesand in each case, approximately 100 μL of the purified poloxamer 407 wasinstilled upstream again. In two animals, a third application afterabout 13 to 14 minutes, of approximately 100 μL was needed to providethe bloodless surgical field. Most of the blood collected during theapplication of the purified poloxamer 407 was deemed to stem from thesnare holes, but blood volume collected was not corrected for this inthe gel experiments as well as the tape experiments. After 15 minutes,the remaining blood was removed, and added to that previously collectedto quantify total arteriotomy blood less for the 15 minutes withpurified poloxamer 407 in place. The order of vessel occlusion wasreversed for two animals with the gel first, followed by the tapes.

The volume of blood captured during the two 15 minute periods, with andwithout internal vessel occlusion with purified poloxamer 407, werecompared and are shown in FIG. 2. The application of purified poloxamer407 reduces bleeding by approximately 90% in the four animals.

The graft was sutured onto the arteriotomy with either the gel presentor the tapes utilized and after two additional hours, fluoroscopy wasused to evaluate patency of the graft. In three pigs in which theevaluation could be performed, the grafts were patent. Euthanasia wasadministered under anesthesia. A sample of subtended myocardium washarvested and sent for histology to assess for myocardial ischemia orearly necrosis. Normal myocardium was found by histological evaluation.

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. patent application publications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of occluding a vascular site in amammal, comprising the steps of: (a) introducing into the vasculature ofa mammal, at or proximal to a surgical site, a composition comprising atleast one purified inverse thermosensitive polymer, wherein said atleast one purified inverse thermosensitive polymer gels in saidvasculature, thereby temporarily occluding said vascular site of saidmammal; and (b) performing an anastomosis.
 2. The method of claim 1,wherein said anastomosis comprises connecting a first vessel and asecond vessel, wherein the anastomosis is end-to-end anastomosis,side-to-end anastomosis, and side-to-side anastomosis.
 3. A method ofoccluding a vascular site in a mammal, comprising the steps of: (a)introducing into the vasculature of a mammal, at or proximal to asurgical site, a composition comprising at least one purified inversethermosensitive polymer, wherein said at least one purified inversethermosensitive polymer gels in said vasculature, thereby temporarilyoccluding said vascular site of said mammal; and (b) performing asurgical procedure comprising anastomosis wherein said anastomosiscontrols blood oozing.
 4. The method of claim 1, wherein said inversethermosensitive polymer is a poloxamer or poloxamine.
 5. The method ofclaim 1, wherein said inverse thermosensitive polymer is poloxamer 407,poloxamer 338, poloxamer 188, poloxamine 1107 or poloxamine
 1307. 6. Amethod of occluding a vascular site in a mammal, comprising the stepsof: (a) introducing into the vasculature of a mammal, at or proximal toa surgical site, a composition comprising at least one purified inversethermosensitive polymer, wherein said at least one purified inversethermosensitive polymer gels in said vasculature, thereby temporarilyoccluding said vascular site of said mammal; and (b) performing ananastomosis, wherein the inverse thermosensitive polymer is poloxamer407, poloxamer 338, poloxamer 188, poloxamine 1107 or poloxamine 1307.